Surface-expressed Toll-like receptors (TLRs) and a newly described class of cytosolic pattern recognition receptors (PRRs), members of the Nod-like receptor (NLR) family, play a major role in innate host defense against the intracellular pathogen Francisella tularensis, a category A biothreat agent. Recently it was shown that F.tularensis triggers TLR2 signaling in immune cells and that mutation of this PRR is associated with greater susceptibility to infection. Similarly, signaling through apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), an NLR adaptor molecule, and subsequent caspase-1 activation were shown to be essential for eliciting protective immunity to F. tularensis infection. One intriguing question which remains unanswered is the precise relationship between the TLR and NLR signaling pathways and whether activation of NLRs in response to F. tularensis occurs downstream of TLRs or proceeds independent of TLR signaling. Furthermore, the specific component(s) of F. tularensis that triggers the downstream signaling events and the receptor(s) of the NLR pathways are not known. This proposal seeks to address these issues by testing the hypothesis that integration of signals transduced via the TLR2 and NLR pathway is required for the optimal development of innate immune responses to F. tularensis infection via the following Specific Aims: 1) Identify the F. tularensis SchuS4 component(s) activating the NLR pathway and evaluate the role of select NLRs in mediating the pro-inflammatory responses to F. tularensis. 2) Establish the key sites of integration between the TLR2 and NLR signaling pathways in response to F. tularensis SchuS4. This proposal will study the cell biology of signal integration between key components of innate immunity and how these processes at the cellular level contribute to the host's ability to resist microbial challenge. Defining the molecular mechanism(s) of innate immunity will be an essential first step towards development of immunotherapeutic, as well as effective vaccine strategies to combat a variety of intracellular bacterial pathogens. This proposal is innovative because it provides a comprehensive approach to explore the relatively unknown signal integration between extra- and intracellular innate immune mechanisms following F. tularensis SchuS4 infection in our CDC-approved ABSL3/BSL3 facility. This study will identify the F. tularensis component(s) and the receptor(s) responsible for triggering the cytosolic machinery and elucidate the molecular mechanisms by which extra- and intracellular signals are integrated to elicit an optimal innate immune response. PUBLIC HEALTH RELEVANCE: This proposal aims to investigate the immunopathologic mechanisms underlying tularemia, a zoonotic disease caused by Francisella tularensis, a Category A biothreat agent. The studies will focus on exploring the cell biology of signal integration between key components of innate immunity and how these processes at the cellular level contribute to the host's ability to resist microbial challenge. Defining the molecular mechanism(s) of innate immunity will be an essential first step towards development of immunotherapeutic, as well as effective vaccine strategies to combat this fatal disease.